Chromosomal rearrangements maintain a polymorphic supergene controlling butterfly mimicry

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S1 .

Figure S1

Published in which should be cited to refer to this work.

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Figure S1. Fine mapping and annotation of the BAC tilepath,. a. Number of recombinants between markers and P, highlighting the 400-Kb zero-recombinant P mapping interval (pink block). b. Overlap of individual BAC clones when aligned to the H. melpomene reference sequence. Clones 38G4, 24I10, and 45B17 are finished and contiguous, but align to the reference sequence in two separate segments due to the rearrangements. The segments are joined on the diagram by a dotted line. Blunt ends are joined together, while round ends represent BAC ends. Red arrows indicate rearrangements breakpoints; grey dots represent shared markers used in sequencing or mapping; coloured dots represent shared markers closest to the breakpoints. c. Names of the markers used for mapping (see Table S6 for marker details). d. Graphical overview of the annotation. Exons of each putative gene are represented by separate bars of the same colour; colour coding is conserved with Fig. 2a. Also see Table S3 for gene details and orthologues

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Figure S2. Graphical overview of H. numata sequence similarity to H.

melpomene. The consensus sequence for the entire P walk, corresponding to BP0 (reference gene order) with the different contigs concatenated, was compared to the HmYb-HmSb sequence using the Shuffle-LAGAN alignment program

¹

and visualised using in VISTA

^2,3

.

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Figure S3. Complete LD between SNPs in perfect association with pattern. The 39 sites in our dataset whose genotypes are in complete association with colour pattern phenotypes (Fig. 2) show complete LD with each other, across a 500-kilobase segment. This segment corresponds to the position of the polymorphic rearrangements associated with mimicry polymorphism in this species, and maintaining divergent haplotypes clades causing the full LD between those associated sites. The

rearrangement order shown is BP1

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Figure S4.a

Figure S4. Comparison of haplotype networks for gene markers within and outside the supergene in Eastern Peru. Haplotype networks were constructed by parsimony using Network (see Methods). For clarity of illustration, networks shown were pre-processed by star contraction

⁴

. Each individual genotype is represented by its two haplotypes (see methods), coloured according to the individual's phenotype.

(a). P-linked markers. Networks for markers within the rearrangements show a very strong haplotype structure, with a minimum of two very divergent (1-4%) haplotypes groups, indicative of suppressed recombination. Markers ERCC-6, LRR and NF-X1, represented here, show a perfect pattern of association with colour pattern genotype:

all homozygous recessive silvana (P

^sil

P

^sil

) or illustris (P

^ill

P

^ill

) individuals, and only

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Figure S4.b

these individuals, are homozygous for one haplogroup, while all aurora (P

^aur

-) individuals and associated phenotypes (elegans, isabellinus and arcuella) harbour at least one haplotype in the alternative haplogroup. This striking two-part structure remains for HN00021, at the boundary of the mapping interval; however, for this marker the association with colour pattern is already largely lost (Fig. 2b), as can be seen by many silvana individuals showing haplotypes in both haplogroups. (b). The networks become mostly unstructured for flanking markers on either side of the supergene (BmSuc, recQ Helicase, NudC). (c). Markers unlinked to P show a clear lack of haplotypes structuring (Bm18W, VAS and AATC shown –see also Table S6 and

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Figure S4,c

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Figure S5. Fst plot of differentiation in a natural population. Fst was computed using the software DNAsp

⁷

to measure the level of genetic differentiation between individuals with the recessive silvana vs. aurora phenotype in the Yurimaguas

population. Because of the allelic frequency of ~0.6 for the recessive P

^sil

allele

⁸

, about half of aurora individuals are expected to be heterozygous for the recessive allele;

therefore the maximum level of Fst lies around 0.5. Note that the very high level of genetic differentiation between the aurora and silvana individuals around the colour- pattern supergene P derives from the lack of recombination at this locus, and decays rapidly for loci outside or unlinked to the supergene.

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Figure S6. Conserved within-population haplotype structure between Peru and French Guiana. Haplotype networks were constructed as presented in Fig. S4 for loci within the supergene (a. ERCC6, b. LRR, c. Rnu3 –see Fig.2), including haplotypes derived from a French Guianan population. The two populations share the same silvana haplotype group (bottom recessive P

^sil

allele, coloured yellow for Peru and

aurora/numata haplogroup

a. HN0040/ERCC6

b.HN0024/LRR

c. HN0046 RNU3

4 substitutions 10 substitutions

7 substitutions

silvana haplogroup

silvana - Peru illustris - Peru aurora - Peru arcuella - Peru numata - FG silvana - FG Phenotypes

(916bp)

(468bp)

(1062bp)

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pale blue for FG), and the dominant haplotype group containing haplotypes for similar morphs aurora and numata (P

^aur

and P

^num

, coloured brown and purple, respectively)

⁶

. Other colours used are orange for arcuella, clustering with the aurora/numata clade, except when heterozygous for a silvana allele. The within-population haplotypes structure as well as the perfect SNP-phenotype association, are conserved despite the 2,900 km separating Peru and French Guiana. This confirms that the network is structured primarily by phenotype across the Amazonian range, and not by population, and that the SNP-phenotype association is not a product of local population processes.

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Table S1. Cross-over rate estimates in P-linked and unlinked regions

Brood` Markers LG distance Total number of individuals Number of recombinants Cross-over rate (Kb) (N) (N^rec) (N^rec/nucleotide)^d Unlinked regions

Brood 472 Ge03 - Ge30B LG17 66.1 60 7 1.76 x 10^-6

Brood 472 TRP - c30739 LG13 67.8 70 5 1.05 x 10^-6

Brood 511 TRP - c30739 LG13 67.8 39 3 1.13 x 10^-6

Brood 502^a TRP - c6511 LG13 52.3 87 8 1.76 x 10^-6

Total unlinked regions 256 23 1.93 x 10^-6

P linked regions

All broods^b B1B - Hn00021 LG15 244.5 366 2 2.23 x 10^-8

All broods^c Hn00021 - LRR LG15 323.5 366 0 <8.45 x 10^-9

All broods^b LRR - 3O10 LG15 321.5 366 4 3.40 x 10^-8

Total P and flanking regions^e 366 6 1.78 x 10^-8

a An alternative marker (c6511) was used due to the absence of paternal variation at the c30739 marker

b Flanking regions of P

c Mapping interval for P

d calculated as the number of crossing-overs per nucleotide using the total length surveyed, in base pairs

e More distant loci mapping to linkage group 15 are at unknown physical distance of our P tilepath. Linkagemapping and recombination distances along this and other linkage groups in H. numata are published elsewhere⁹.

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Table S2. BAC clone data and accessions

Clone Accession Size (bp) Overlap

6M17 FP476061 121000 24I10, 49B1, 20J7, 34H10 7C9 FP565803 115981 8K22, 38G16 14K13 CU856181 96570 46M23, 38G4

24I10 CU856182 110815 45B17, 24I10, 49B1, 6M17 31F4 CU655868 95876 41G24, 38C19

35N20 FP885880 108899 23N8 36N20 FP885855 95024 43D2, 34P21 38C19 CU914733 86858 41G24, 31F4

38G4 CU655869 140835 46M23, 14K13, 45B17, 24I10 41G24 CU914734 109797 38C19, 31F4

43D2 CU633161 131291 36N20, 34P21 45B17 CU638865 90190 38G4, 24I10, 49B1 46M23 CU856175 100298 38G4, 14K13

49A1 FP884220 91922 unlinked (on chromosome 17) 49B1 FP236755 96772 45B17, 24I10, 6M17, 20J7 34H10^‡ FP885879 141031 20J7, 6M17

20J7 FP885878 108308 24I10, 49B1, 6M17, 34H10 1K7 FP885863 118130 -

8K22 FP476023 101778 8K22, 7C9 20L19 FP476047 82043 unlinked 23N8 FP885857 102695 35N20 34P21 FP885861 62129 43D2, 36N20 38G16 FP475989 78536 7C9, 38G16

‡Clones sequences below the line overlap redundantly with other clones

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Table S3. Annotation of the P region.

Gene

number Putative gene name

Best BLASTx

D. melanogaster hit % identity B. mori ortholog % identity Hm ortholog^$ Hn Clone HN00101 Cht7, chitinase-like CG1869 / Cht7 53% BGIBMGA005539 36% - 43D2

HN00102 Diaphanous FH3 CG13913 / mwh 32% BGIBMGA005540 47% - 43D2

HN00103 Rho GDP-dissociation inhibitor CG7823 / RhoGDI 68% BGIBMGA005672 95% - 43D2 HN00104 Guanylyl cyclase CG33114 / Gyc32E 59% BGIBMGA005671 78% - 36N20 HN00105 Type II geranylgeranyl transferase CG18627 / betaggt 50% BGIBMGA005544 69% - 41G24 HN00106 Fork head transcription factor CG11799 / mnf 39% BGIBMGA005669 60% - 41G24 HN00001 Peptidase S9 (truncated?) no hit - BGIBMGA005667 41% HM00001 41G24

HN00002 Peptidase S9 no hit - BGIBMGA005667 47% HM00002 41G24

HN00003 CG10949 CG10949 31% BGIBMGA005547 69% HM00003 41G24

HN00004 Trehalase CG9364 / Treh 32% BGIBMGA005665 69% HM00004 ^[HmYb] 41G24 HN00006 Trehalase CG9364 / Treh 41% BGIBMGA005664 60% HM00006 ^[HmYb] 41G24

HN00007 B9 protein CG14870 33% BGIBMGA005663 59% HM00007 ^[HmYb] 41G24

HN00008 CG5098 CG5098 75% BGIBMGA005548 65% HM00008 ^[HmYb] 41G24

HN00012 CG2519 CG2519 45% BGIBMGA005549 47% HM00012 ^[HmYb] 31F4

HN00013 unkempt CG4620 / unk 86% BGIBMGA005660 70% HM00013 ^[HmYb] 31F4 HN00014 Histone H3 CG8989 / His3.3B 100% BGIBMGA005550 100% HM00014 ^[HmYb] 31F4

HN00017 RecQ Helicase CG6920 / mus309 40% BGIBMGA005666 61% HM00017 ^[HmYb] 31F4

HN00018 Phox-like no hit - BGIBMGA005553 71% HM00018 ^[HmYb] 31F4

HN00107 little imaginal discs CG9088 / lid 46% BGIBMGA005554 31% - 31F4 HN00019 BmSuc2 beta-fructofuranosidase no hit - BGIBMGA005555 55% HM00019 ^[HmYb] 31F4

HN00020 Peptidase M28 CG5976 55% BGIBMGA005556 70% HM00020 ^[HmYb] 31F4

HN00040* ERCC-6 CG4261 / Hel89B 34% BGIBMGA005640 63% HM00040 7C9

HN00039* Licorne MAP kinase CG12244 / licorne 67% BGIBMGA005641 89% HM00039 7C9 HN00038* lethal (2) k05819 CG3054 / lethal (2) k05819 45% BGIBMGA005562 61% HM00038 7C9

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HN00035* domeless, tyrosine phosphatase CG14226 / dome 29% BGIBMGA005642 56% HM00035 7C9 HN00034* F-box/WD-repeat protein pof1 no hit - BGIBMGA005645 51% HM00034 7C9 HN00033* Protein-kinase-like CG32580 / mucin 14A 20% BGIBMGA005561 44% HM00033 ^[HmSb] 7C9 HN00028*† Putative Manf CG7013 / Manf 80% BGIBMGA005559 79% HM00028 ^[HmSb] gap HN00030*† Proteasome 54kD subunit CG7619 / Pros54 61% BGIBMGA005560 79% HM00030 ^[HmSb] gap HN00025* Fizzy CG4274 / Fzy 25% BGIBMGA005652 52% HM00025 ^[HmYb] 38G4

HN00108* Sprint CG34414 / Spri 40% BGIBMGA005653 85% - 38G4

HN00109* CG14470 CG14470 26% BGIBMGA005654 68% - 38G4

HN00110* HN00110 no hit - BGIBMGA005558 69% - 38G4

HN00024* LRR CG5407 / Sur-8 25% BGIBMGA005655 39% HM00024 ^[HmYb] 38G4

HN00023* HN00023 CG10581 42% BGIBMGA005557 52% HM00023 ^[HmYb] 38G4

HN00041* penguin CG1685 / pen 35% BGIBMGA005638 62% HM00041 24I10

HN00042* Thymidylate kinase CG5757 34% no hit - HM00042 24I10

HN00043* CG9414 CG9414 / Drep-4 34% BGIBMGA005639 75% HM00043 24I10

HN00044* RRS1 CG32409 55% BGIBMGA005564 67% HM00044 24I10

HN00045* CG12659 CG12659 55% BGIBMGA005565 81% HM00045 24I10

HN00046* U3 snoRNA CG33505 / U3-55K 50% BGIBMGA005636 78% HM00046 24I10 HN00047* Sr protein CG5442 / SC35 83% BGIBMGA005635 86% HM00047 24I10

HN00049* HN00049 CG15040 18% BGIBMGA005566 33% HM00049 24I10

HN00050* Shuttle-craft CG3647 / stc 36% BGIBMGA005634 46% HM00050 24I10

HN00051* HN00051 CG6793 22% BGIBMGA005567 64% HM00051 24I10

HN00052* Thrombospondin no hit - BGIBMGA005633 52% HM00052 24I10

HN00053* lethal (2) giant larvae CG2671 / l(2)gl 44% BGIBMGA005570 83% HM00053 6M17, 38G4

HN00112 HN00112 no hit - BGIBMGA008616 50% 6M17

HN00054 Zn finger protein CG10366 28% BGIBMGA005571 40% HM00054 6M17

HN00055 Prefoldin CG41265 27% BGIBMGA005632 66% HM00055 6M17

HN00056 Tetratricopeptide repeat protein CG4525 51% BGIBMGA005572 69% HM00056 6M17 HN00057 Putative Ecdysone oxidase CG9512 33% BGIBMGA005711 41% HM00057 6M17 HN00058 cuticular protein RR-2 motif 74 CG15008 / Cpr64Ac 68% BGIBMGA005631 83% HM00058 6M17

HN00059 CG6734 CG6734 / 43% BGIBMGA005629 59% HM00059 34H10

HN00060 rudimentary-like CG3593 / rudimentary-like 51% BGIBMGA005628 71% HM00060 34H10

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HN00116 gawky CG31992 / gw 44% BGIBMGA005589 73% - 35N20

HN00117 CG5262 CG5262 53% BGIBMGA005613 73% - 35N20

HN00118 CG10254 CG10254 55% BGIBMGA005588 81% - 35N20

HN00119 RabGAP Regulator CG5337 58% BGIBMGA005614 83% - 35N20

HN00120 Alcohol dehydrogenase CG17221 28% BGIBMGA005615 67% - 35N20

HN00121 putative NudC CG31251 32% BGIBMGA005586 61% - 35N20

HN00122 CG6398 CG6398 46% BGIBMGA005617 - 35N20

* Gene in a rearranged chromosomal segment

$ Superscripts indicate genes in HmYb or HmSb interval in H. melpomene

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Table S4. Sequence similarity to H. melpomene HmYb-HmSb region

Clone^† Conserved Conserved length (bp)

6M17 84% 101605

7C9 75% 87429

14K13 69% 66528

20J7 84% 46930

24I10^‡ 88% 89057

31F4 87% 83666

34H10 84% 118293

36N20 79% 74983

38C19 84% 73050

38G4^‡ 50% 65467

41G24 85% 93781

43D2 80% 87688

49B1 81% 27499

Consensus P walk 75% 649756

† Redundant clones omitted

‡ Clone with a rearrangement breakpoint: calculation based on longer side of breakpoint. Note that the longer side of breakpoint BP1 is shorter in clone 45B17 than in 24I10, so the latter is shown.

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Table S5. List of specimens from natural populations

Code H. numata form Sex Location Latitude Longitude Country

b001-MJ05 bicoloratus female Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru b004-MJ05 bicoloratus male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru b005-MJ05 bicoloratus male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru b007-MJ05 bicoloratus male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru b011-MJ05 bicoloratus female Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru b012-MJ05 bicoloratus female Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru b122-MJ05 bicoloratus male Biodiversidad km19 Tarapoto-Yurimaguas 6°27'40.47"S 76°17'7.04"W Peru t003-m05 tarapotensis female Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru t008-m05 tarapotensis male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru t036-m05 tarapotensis male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t057-m05 tarapotensis male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru t058-m05 tarapotensis male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru t060-m05 tarapotensis male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru t063-m05 tarapotensis male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru t1328-05 tarapotensis male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru o1273_05 aurora male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru o1275_05 aurora female km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru o140_m05 aurora (x arcuella) female km 7.2 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru o207_m05 aurora female km 6.5 Pongo-Barranquita 6°17'51.12"S 76°14'3.96"W Peru e1277_05 aurora (var. elegans) female km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru e139_m05 aurora (var. elegans) female km 7.2 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru L1266_05 aurora (var. isabellinus) male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru L1270_05 aurora (var. isabellinus) male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru s089_m99 silvana male km 7.2 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru s093_m99 silvana male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru s094_m99 silvana male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru s095_m99 silvana male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru s096_m99 silvana male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru s097_m99 silvana male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru

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Additional specimens for Association and LD studies (fig. 2)

a044_m99 arcuella male km 7.2 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru a077_m99 arcuella female Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru a1274_05 arcuella male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru a202_m05 arcuella male km 6.5 Pongo-Barranquita 6°17'51.12"S 76°14'3.96"W Peru o076_m99 aurora female Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru o1206_05 aurora female km 6 Pongo-Barranquita 6°17'51.12"S 76°14'3.96"W Peru o1207_05 aurora female km 6 Pongo-Barranquita 6°17'51.12"S 76°14'3.96"W Peru o1244_05 aurora female km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru o1264_05 aurora male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru o1269_05 aurora male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru o1272_05 aurora male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru o129_m02 aurora female km 7.5 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru o132_m99 aurora female km 7.2 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru o149_m99 aurora female Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru o203_m05 aurora male km 6.5 Pongo-Barranquita 6°17'51.12"S 76°14'3.96"W Peru o206_m05 aurora male km 6.5 Pongo-Barranquita 6°17'51.12"S 76°14'3.96"W Peru o215_m02 aurora male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru o253_M02 aurora male km 7.5 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru o256_m02 aurora female km 7.5 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru o293_m02 aurora male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru o294_m02 aurora male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru o1265_05 aurora female km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru o1267_05 aurora male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru o208_m05 aurora female km 6.5 Pongo-Barranquita 6°17'51.12"S 76°14'3.96"W Peru o1268_05 aurora (var.) male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru g078_m99 aurora (var. isabellinus) male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru s124_m05 silvana male Biodiversidad km19 Tarapoto-Yurimaguas 6°27'40.47"S 76°17'7.04"W Peru s1240_05 silvana male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru s1241_05 silvana female km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru s1242_05 silvana male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru

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s132_M02 silvana female km 7.5 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru s183_m05 silvana male Alianza km 80 Tarapoto-Yurimaguas 6°7'54.51"S 76°16'7.33"W Peru s184_m05 silvana male Alianza km 80 Tarapoto-Yurimaguas 6°7'54.51"S 76°16'7.33"W Peru s205_m05 silvana male km 6.5 Pongo-Barranquita 6°17'51.12"S 76°14'3.96"W Peru s219_m02 silvana male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru s221_M02 silvana female Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru s247_M02 silvana male km 7.5 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru s472_m02 silvana female Ex larva Pintoyaquillo 5 km NW Convento 6°15'05.04"S 76°18'48.96"W Peru s547_m02 silvana male Villa Autonoma, Tarapoto 6°28'43.07"S 76°21'18.60"W Peru Additional specimens for Haplotype networks and Short-fragment Breakpoint assays (fig. S4, S5)

a1243-05 arcuella female km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru o100_M99 aurora male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru o102_M99 aurora male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru o104_M99 aurora female Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru o105_M99 aurora female Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru o228_M02 aurora male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru o229_M02 aurora male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru o043_M99 aurora female km 7.2 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru o064_M99 aurora female Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru o075_M99 aurora female Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru o079_M99 aurora male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru o080_M99 aurora male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru o081_M99 aurora male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru o082_M99 aurora male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru o084_M99 aurora male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru o085_M99 aurora male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru o090_M99 aurora male km 7.2 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru o098_M99 aurora male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru o099_M99 aurora male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru o217-m02 aurora male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru o227-m02 aurora male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru o106-m99 aurora male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru e421_M02 aurora (var. elegans) male Ex larva km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru

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g106_M99 aurora (var. isabellinus) male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru g083_M99 aurora (var. isabellinus) male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru s168_M97 silvana male Túnel km 20 Tarapoto-Yurimaguas 6°27'2.67"S 76°17'26.47"W Peru s178_M99 silvana male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru s067_M99 silvana female Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru s068_M99 silvana male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru s069_M99 silvana male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru s070_M99 silvana male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru s071_M99 silvana male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru s072_M99 silvana male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru s073_M99 silvana male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru s087_M99 silvana male km 7.2 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru s088_M99 silvana male km 7.2 Pongo-Barranquita 6°17'22.02"S 76°13'43.74"W Peru s091_M02 silvana male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru s092_M99 silvana male Pongo de Cainarachi km 60 TY 6°20'30.11"S 76°17'52.98"W Peru s155-m99 silvana male km 10 Tarapoto-Yurimaguas 6°27'43.00"S 76°19'23.00"W Peru s64b-m99 silvana female Davidcillo km 72 Tarapoto-Yurimaguas 6°14'55.00"S 76°15'50.00"W Peru i086_M99 illustris male Davidcillo km 72 Tarapoto-Yurimaguas 6°14'24.50"S 76°16'12.84"W Peru i204_M05 illustris male km 6.5 Pongo-Barranquita 6°17'51.12"S 76°14'3.96"W Peru i1189_05 illustris male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru i121_M05 illustris female Biodiversidad km19 Tarapoto-Yurimaguas 6°27'40.47"S 76°17'7.04"W Peru i1276_05 illustris male km 26 Yurimaguas-Tarapoto 5°58'29.34"S 76°13'51.36"W Peru i153_M97 illustris male km 10 Tarapoto-Yurimaguas 6°27'43.00"S 76°19'23.00"W Peru i155_M97 illustris male km 10 Tarapoto-Yurimaguas 6°27'43.00"S 76°19'23.00"W Peru i161_M02 illustris female La Antena km 17 Tarapoto-Yurimaguas 6°27'10.08"S 76°17'55.32"W Peru i166_M97 illustris male Túnel km 20 Tarapoto-Yurimaguas 6°27'2.67"S 76°17'26.47"W Peru i1359-05 illustris male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru b006-MJ05 bicoloratus male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru b028-MJ05 bicoloratus male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru b29-m05 bicoloratus male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru b30-m05 bicoloratus female Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru

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b61-m05 bicoloratus male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru b62-m05 bicoloratus male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru b123-m05 bicoloratus Fundo Biodiversidad km19 Tarapoto-Yurimaguas 6°27'40.47"S 76°17'7.04"W Peru b125-m05 bicoloratus Fundo Biodiversidad km19 Tarapoto-Yurimaguas 6°27'40.47"S 76°17'7.04"W Peru b126-m05 bicoloratus Fundo Biodiversidad km19 Tarapoto-Yurimaguas 6°27'40.47"S 76°17'7.04"W Peru b127-m05 bicoloratus Fundo Biodiversidad km19 Tarapoto-Yurimaguas 6°27'40.47"S 76°17'7.04"W Peru b64-m05 bicoloratus male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru b79-m05 bicoloratus female Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru b86-m05 bicoloratus male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru b140-m02 bicoloratus male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru b150-m02 bicoloratus male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru b212-m02 bicoloratus male km 10-12 Tarapoto-Yurimaguas 6°27'43.00"S 76°19'23.00"W Peru b263-m02 bicoloratus female Puente Rio Serranoyacu Rioja 5°40'28.85"S 77°40'10.88"W Peru b267-m02 bicoloratus female Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru b282-m02 bicoloratus male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru b283-m02 bicoloratus male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru b731-m02 bicoloratus male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru ba73-m97 bicoloratus male Cataratas del Ahuashiyacu km14 TY 6°27'15.60"S 76°18'24.99"W Peru t1187-05 tarapotensis male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru t1188-05 tarapotensis male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru t1191-05 tarapotensis male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru t1327-05 tarapotensis male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t1358-05 tarapotensis female Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru t1360-05 tarapotensis female Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t1361-05 tarapotensis female Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t1362-05 tarapotensis male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t1363-05 tarapotensis male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t1365-05 tarapotensis female Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t50-m97 tarapotensis female Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t53-m97 tarapotensis male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t56-m97 tarapotensis male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t69-m97 tarapotensis male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t71-m97 tarapotensis female Cataratas del Ahuashiyacu km14 TY 6°27'15.60"S 76°18'24.99"W Peru

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t176-m02 tarapotensis male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t203-m02 tarapotensis female Shapaja Quebrada Pucayaquillo 6°35'10.00"S 76°13'5.00"W Peru t206-m02 tarapotensis female Chumía, Chazuta 6°37'1.77"S 76°11'4.55"W Peru t207-m02 tarapotensis female Chumía, Chazuta 6°37'1.77"S 76°11'4.55"W Peru t211-m02 tarapotensis male km 10-12 Tarapoto-Yurimaguas 6°27'43.00"S 76°19'23.00"W Peru t268-m02 tarapotensis female Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t270-m02 tarapotensis male Fundo Biodiversidad km19 Tarapoto-Yurimaguas 6°27'40.47"S 76°17'7.04"W Peru t272-m02 tarapotensis male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru t284-m02 tarapotensis male Rio Shilcayo, Tarapoto 6°27'20.00"S 76°20'40.00"W Peru a1192-05 arcuella male Urahuasha km 8 Tarapoto-Yurimaguas 6°27'50.11"S 76°19'51.30"W Peru u131-m99 arcuella (var. lutea) male Shapaja Quebrada Pucayaquillo 6°35'10.00"S 76°13'5.00"W Peru n4123-M09 numata male Les Nouragues Inselberg 4°5'0.10" 52°40'31.46" FG n4124-M09 numata female Les Nouragues Inselberg 4°5'0.10" 52°40'31.46" FG n4183-M09 numata female Les Nouragues Inselberg 4°5'0.10" 52°40'31.46" FG n4111-M09 numata male Les Nouragues Inselberg 4°5'0.10" 52°40'31.46" FG n4112-M09 numata male Les Nouragues Inselberg 4°5'0.10" 52°40'31.46" FG n4125-M09 numata male Les Nouragues Inselberg 4°5'0.10" 52°40'31.46" FG n4181-M09 numata female Les Nouragues Inselberg 4°5'0.10" 52°40'31.46" FG n4182-M09 numata male Les Nouragues Inselberg 4°5'0.10" 52°40'31.46" FG n4196-M09 numata female Les Nouragues Inselberg 4°5'0.10" 52°40'31.46" FG n4146-M09 numata female Les Nouragues-Saut Pararé 4°1'50.75" 52°40'57.31" FG n4147-M09 numata female Les Nouragues-Saut Pararé 4°1'50.75" 52°40'57.31" FG n4143-M09 numata female Les Nouragues-Saut Pararé 4°1'50.75" 52°40'57.31" FG n4144-M09 numata female Les Nouragues-Saut Pararé 4°1'50.75" 52°40'57.31" FG n4145-M09 numata female Les Nouragues-Saut Pararé 4°1'50.75" 52°40'57.31" FG n4148-M09 numata male Les Nouragues-Saut Pararé 4°1'50.75" 52°40'57.31" FG n4149-M09 numata male Les Nouragues-Saut Pararé 4°1'50.75" 52°40'57.31" FG n4150-M09 numata male Les Nouragues-Saut Pararé 4°1'50.75" 52°40'57.31" FG n4152-M09 numata male Les Nouragues-Saut Pararé 4°1'50.75" 52°40'57.31" FG n4153-M09 numata female Les Nouragues-Saut Pararé 4°1'50.75" 52°40'57.31" FG n4154-M09 numata female Les Nouragues-Saut Pararé 4°1'50.75" 52°40'57.31" FG

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n4021-M09 numata male Patawa Laie#3 4°33'43.91" 52° 9'24.99" FG n4022-M09 numata male Patawa Laie#3 4°33'43.91" 52° 9'24.99" FG n4051-M09 numata female Patawa Laie#3 4°33'43.91" 52° 9'24.99" FG n4053-M09 numata female Patawa Laie#3 4°33'43.91" 52° 9'24.99" FG n4054-M09 numata male Patawa Laie#3 4°33'43.91" 52° 9'24.99" FG n4225-M09 numata female Route de Cacao, Montagne Grosse Roche 4°33'52.14" 52°25'36.74" FG n4226-M09 numata female Route de Cacao, Montagne Grosse Roche 4°33'52.14" 52°25'36.74" FG n4223-M09 numata female Route de Cacao, Montagne Grosse Roche 4°33'52.14" 52°25'36.74" FG n4224-M09 numata female Route de Cacao, Montagne Grosse Roche 4°33'52.14" 52°25'36.74" FG

n4241-M09 numata female Route de Kaw, Escol+2km FG

n4017-M09 numata female Route de Regina, Morne aux Echos 4°38'9.45" 52°21'38.52" FG s4122-M09 silvana female Les Nouragues Inselberg 4°5'0.10" 52°40'31.46" FG s4184-M09 silvana male Les Nouragues Inselberg 4°5'0.10" 52°40'31.46" FG s4151-M09 silvana female Les Nouragues-Saut Pararé 4°1'50.75" 52°40'57.31" FG s4025-M09 silvana female Patawa Laie#3 4°33'43.91" 52° 9'24.99" FG s4010-M09 silvana female Route de Regina, Morne aux Echos 4°38'9.45" 52°21'38.52" FG

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Table S6. Short-range PCR breakpoint assay.

Positive assays

Population Morph N BP1 BP2 BP0

Yurimaguas, Peru silvana 34 34 (100%)

(n=82) aurora 42 42 (100%)

^a

28 arcuella 6 6 (100%) 4

Tarapoto, Peru illustris 8 8 (100%)

(n=74) tarapotensis 34 34 (100%) 12

bicoloratus 32 30 (94%) 17 9

French Guiana silvana 5 5 (100%)

(n=40) numata 35 35 (100%) 28

aPercentages indicate the proportion of positive assays for the breakpoint associated with the expressed phenotype.

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Table S7. Breakpoint primer pairs

Breakpoint Clone Expected product size (bp) Gene name Primer Sequence

BP0-Long range bHN7C9 5609 HN00040 TTGCAATTTTAATCGCTTCC

bHN45B17 HN00041 GTTAGTGCCCTGCCAAACAC

BP0-short range bHN7C9 1100-1300^a HN00040 TGCAATAGCCAATTGAGTCT

bHN45B17 HN00041 TCAAATACTTCCRCAGTAGC

BP1-Long range bHN45B17 2592 HN00041 GTTAGTGCCCTGCCAAACAC

bHN38G4 HN00023 CCATTTTGCCAATTTCGTCT

BP1-short range bHN24I10 457 na GCAAAATCCCTTGAGAGCTG

bHN24I10 na TTCCCGCATTTTATTTCGTC

BP2-Long range bHN6M17, 38G4 2432 HN00053 CCTATCGACGCATTTTATTC

bHN38G4 HN00023 CCATTTTGCCAATTTCGTCT

BP2-short range bHN38G4 324 na GCGGCTAGCACTTAAACAGC

bHN38G4 na TCGGTCTCGGTCAAAAAGTC

a polymorphic fragment length

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Table S8. Marker and primer details

Marker name Clone Estimated walk Length Gene name^‡ Forward primer Reverse primer position (Kb)^† (bp)

P-linked markers

B1B 43D2 115929 514 intergenic GTAGGGATTTTAGGTTATGGTTTT AACATATTATCGGCTTCGTCA GuaCycl 43D2 121879 655 HN00104 GCGACGCCATCATATCCTAT CGTATATTGGGCACGGAATC

Fox 41G24 195995 681 HN00106 CTGCAGATTGGGCTGATTTT CATTCGATTTCAACGGTGTG

a41 41G24 201268 357 intergenic ATCGTCGAACTCCTGACCAC TTTTGTTCCACTATTACGCAAAAA

B1A 41G24 206838 410 intergenic CTACTACTGCGCTACAAAGGTC TGAGTAGAGATGCCAAAAAGTA Trehalase1A 41G24 246620 710 HN00006 AGCCCTGAACGTAAGCAAGA ACATGAGCACCCTCAACTCC

CG14870 41G24 266045 772 HN00007 TGCGAGAATCTGGAGTAACAAA GGTCTACCAGCTCTGGATGC CG18292 41G24 289368 251 HN00011 TCAGTGATATGGAAGCGATGGAT AATGCCTCTTTTTAGTCTTTCTGC CG2519in5 31F4 302294 661 HN00012 TTACTGGTGTTCCCGTTGGT GTGACGCAGAGGCACAGTTA

unk-in1 31F4 320341 622 HN00013 TCCAGATGGAACTGATGGTG GGCAGGCATACCCTTGTCTA

recQ 31F4 333478 736 HN00017 CGGGCAGCATTCTCAATTAT TGTTCTCAGTGGGGACATGA

invertase/BmSuc 31F4 346506 918 HN00019 AATTGGGGACACGCTGTAAG AATACGCACGCCTAATACCG KG17-11j7 31F4 351529 187 HN00020 CAACATTAAGAATGGGGTAGGAG TGCTTACCTTTTCTTGATAGAATTT

CG5976 31F4 353577 721 HN00020 TAAATCCCGATGCCGATAGA CACCGCCGAGTCTGTAGC

CG5976in2 31F4 353577 921 HN00020 CACATCAAACGCCGTAATTG AGTTGTCACGAGACGTGTGC Hm21ex6 gap 360475 601 HN00021 AAAAATCAAGGAAGAATAAAGATGC TCCAGCAAAGATCCTTTGAAC EnoyalCoAin2 gap 365475 832 HN00022 TCAGGAGGCTGAGAAGATGG GAAGACGCGGCTTACAAGA ERCC6-F15 7C9 370545 947 HN00040 ATCACATGGCGGAAATCATC GCTGACCAATTCTCCATGCT

16D20-sp6 7C9 395721 519 intergenic GTTCGGCACACCATATCCTC GAGTTCAATCCGCCATTTGT 30F8-sp6 7C9 442278 529 HN00035 TTGCTGTGTATCCACTTGGTG ATATCTCGGTTGGCTGCTTG

Wdr13 7C9 451148 250 HN00034 CTCCTGAGTTCGGTGGAAAG CCAGTAACCGGTTCGCTATG

16D20-T7 7C9 470956 851 intergenic ATCTGATGCGAAGGAAGTGG GGGATGTCGACGCTAGAAAA

Pros54 gap 521456 756 Hm00030 CAACAAGACTGCAGGCACAG TTCAGCCTGTGGAGCTTCTT

ARP-like gap 526456 434 Hm00028 TGGTGTGTTTTATTTATTATTCCTGGT TCAGATCTACCCATATATCTAGGTTTT

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ATP-Bm5558 38G4 687036 244 HN00023 AGGCTTTTGACCGAGGAAGT CCATTTTGCCAATTTCGTCT XTP5in2 45B17 693662 364 HN00041 GTTAGTGCCCTGCCAAACAC TGCAAGGAATTGCACATTTT

RnU3 45B17 712951 1062 HN00046 CGCTCCTAACCTCGAAAATG GTGCTCCTGCCCTATAGCTG

NF-X1 45B17 736354 1136 HN00050 CCTCCTAATAATTGGAATGGTGA CCACAGACGATTTATGTCTTCC Thrombosp. 45B17 754236 656 HN00052 ACCTGTGGACCTGGTAGACG CCACTACCACACCAGGCTCT 31J7-sp6 34H10 932147 206 intergenic TCCAAGTGAATCCCCAAGAG ATCAGGCACGGTCTTCGATTA

3O10 gap 1005448 320 intergenic ACCCCAGGTTTCGGAAATAA CAGGGTGTGACGACGATAAA

GCP/Bm5536 gap 1124127 557 BM05536 CACGTCAATGAGATGGCAGT AACTATGCACCCCTGAAGCA NudC/Bm5586 23N8 1351299 671 HN00121 TATTCAATTTTCTCGCAAGG CCCTCACTGATACTCCAGAA Srp68/Bm5593 1K7 1946672 854 BM05593 CGTCGTGTCACAGTGCGCCC TACTTGTCACTCTTGCCCCA Unlinked markers

Bm18W unlinked LG18, BmCh23 1274 BM11038 TAGGGTCGTAATTCTGGGTC AAACAATATGACTGCCACCA LAM unlinked LG17, BmCh13 863 BM00910 TCCAGAATGCTATGCTTGTG TTAATTCAGATGTAAGCTCT Sumo unlinked (not mapped) 678 BM02755 CCAAATCCGCTTATGG GAAGAAAAACATGTTATTAT TL unlinked LG09, BmCh07 660 BM10185 GATGCCACGTCCGTTAGAGA CTATGAGCTTGTTGAATACC VAS unlinked LG13, BmCh22 639 BM00231 ATCACAAGACTTCTCCGTTT TTTTCTTCTTAAGTTACTGG AATC unlinked LG18, BmCh23 795 BM11029 TGATTTTAAGCTGCACAAGGA ACTTACAATTTTTCAATCAT CDP unlinked LG12, BmCh08 747 BM05460 AACAAAATGGGAAAAACATC TCCTTGTATGGGGGATTGAT CAT unlinked LG21, BmCh01 1039 BM00701 TCAAGACTGCGATTCAAACA TGTCTTCAGTTTGTCCACT CMD unlinked LG19, BmCh12 825 BM10423 CTTTTGTATCAAATTGCATCT AATCTTTACAAACATCTAG HCL unlinked LG18, BmCh23 706 BM11272 GCCGTAAAAGCAACCAC AACATATAAATTACACCAAA Hsp90 unlinked BmCh27 980 BM04612 AAATGCCAGAGGAAAAAATG GATAAGGTCTTCACAGTTGT ATPs unlinked LG06, BmCh09 1034 BM12555 GCAGGAAAAGGCCAAGG TCCATACCCAAGATGCAAT Cna unlinked LG20, BmCh10 1116 BM06850 GTGCCACCTTATTACGAT TGGTGGCATCCTTTCATTT TRP_exon1-2 unlinked LG13, BmCh14 473 BM09272 GAGGACGACGTGATGGAGAT CCGATTTGGAAGTCCTTGAG c30739_exon3-4 unlinked LG13, BmCh14 514 BM09230 CGCATCTAGCCACAGATGAA GATAGGGGTTCGGATGGTTT c6511_exon3-4 unlinked LG13, BmCh14 583 BM09232 TCAATTTGACCAACCACTCG TAAATCGAACGGCGTACCTC 20L19_Ge03 unlinked LG17, BmCh13 634 intergenic AAATCCATGAACTTGGTTTACAA TGAGAAACCGGTTGACTTGG 20L19_Ge30B unlinked LG17, BmCh27 525 BM04620 AACGCTAAACGGGCGGTA TATCATTTTGTGTATTTTGAGGATTTT

† Estimated position (in kilobases) along the walk taking into account the hypothesised length of the gaps, based on the H. melpomene HmYb-HmSb walk. Unlinked markers

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Table S9. NCBI Accession numbers for population sets

Region Gene Marker n seq Accessions

Peru Acetoacetyl-CoA thiolase (Aact) gene 42 JN174613 - JN174654 Peru Putative ARP-like (HM00028) 41 JN174802 - JN174842 Peru Putative ATPbinding protein (Bm5558, HN00023) gene 34 JN173999 - JN174032 Peru H+ transporting ATP synthase beta subunit (ATPs) gene 38 JN175124 - JN175161 Peru 8 wheeler gene (Bm18W) 37 JN174182 - JN174218 Peru Catalase (CAT) gene 35 JN174074 - JN174108 Peru Cell division protein (CDP/FtsJ) gene 40 JN174655 - JN174694 Peru Cytosolic malate dehydrogenase (CMD/ MDH) gene 25 JN174109 - JN174133 Peru Calcineurin A (cna) gene 41 JN174033 - JN174073 Peru Putative DNA excision repair protein ERCC-6 51 JN173948 - JN173998 Peru Forkhead protein (Fox, FH) gene 53 JN174295 - JN174347 Peru Putative Guanylate (GCP) cyclase 40 JN174843 - JN174882 Peru Hairy cell leukemia (HCL) gene 36 JN174348 - JN174383 Peru Heat shock protein (Hsp90) gene 35 JN174384 - JN174418 Peru Hypothetical protein (HM00021) gene 96 JN173852 - JN173947 Peru Putative Invertase (BmSuc) gene 62 JN174883 - JN174944

Peru Laminin (LM) gene 29 JN174773 - JN174801

Peru Leucine-rich repeat protein (LRR) gene 95 JN174945 - JN175039 Peru Putative Shuttle craft (NFX1) 46 JN175078 - JN175123 Peru Putative Nuclear distribution protein NUDC 41 JN174695 - JN174735 Peru 26S proteasome non-ATPase subunit 4 (Pros54/ Psmd4) gene 40 JN174219 - JN174258 Peru Putative U3 small nucleolar RNA-interacting protein 2 (RNU) 39 JN174419 - JN174457 Peru Helicase (RecQ) gene 45 JN173798 - JN173851 Peru Putative signal recognition particle (Srp68) gene 37 JN174736 - JN174772 Peru SUMO-1 activating enzyme (SAE1) gene 34 JN174458 - JN174491 Peru Trachealess (TL/ Trh) gene 41 JN174532 - JN174572 Peru Trehalase 1a (treh-1a) gene 40 JN174492 - JN174531 Peru Putative Penguin (XTP5) 40 JN174573 - JN174612 Peru ATP synthase 21 kDa proteolipid subunit (VAS) gene 36 JN174259 - JN174294 FG Putative DNA excision repair protein ERCC-6 38 JN175040 - JN175077 FG Leucine-rich repeat protein (LRR) gene 14 JN174134 - JN174147 FG Putative U3 small nucleolar RNA-interacting protein 2 (RNU) 34 JN174148 - JN174181

*FG=French Guiana

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Supplementary References

1 Brudno, M. et al. Glocal Alignment: Finding Rearrangements During Alignment. Bioinformatics 19S1, i62, (2003).

2 Frazer, K. A., Pachter, L., Poliakov, A., Rubin, E. M. & Dubchak, I. VISTA:

computational tools for comparative genomics. Nucl. Acids Res. 32, W273- W279, (2004).

3 Mayor, C. et al. VISTA: Visualizing Global DNA Sequence Alignments of Arbitrary Length. Bioinformatics 16, 1046, (2000).

4 Forster, P., Torroni, A., Renfrew, C. & Rohl, A. Phylogenetic star contraction applied to Asian and Papuan mtDNA evolution. Mol. Biol. Evol. 18, 1864- 1881, (2001).

5 Salazar, C., Jiggins, C. D., Taylor, J. E., Kronforst, M. R. & Linares, M. Gene flow and the genealogical history of Heliconius heurippa. BMC Evol. Biol. 8, (2008).